scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

BioTechniques ◽  
2021 ◽  
Author(s):  
Justin P Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna ◽  
Community Profiling

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

scholarly journals A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

2020 ◽  
Author(s):  
Justin P. Shaffer ◽  
Clarisse Marotz ◽  
Pedro Belda-Ferre ◽  
Cameron Martino ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Sequence Data ◽  
Rna Virus ◽  
Microbial Community Composition ◽  
Rna Extraction ◽  
Acid Extraction ◽  
Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

scholarly journals Microbial community composition in Brazilian stored diesel fuel of varying sulfur content, using high-throughput sequencing

Fuel ◽  
2017 ◽  
Vol 189 ◽  
pp. 340-349 ◽  
Author(s):  
Aline Oliboni de Azambuja ◽  
Francielle Bücker ◽  
Patrícia Dörr de Quadros ◽  
Kateryna Zhalnina ◽  
Raquel Dias ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Sulfur Content ◽  
High Throughput ◽  
Diesel Fuel ◽  
High Throughput Sequencing ◽  
Microbial Community Composition

scholarly journals The Tolerance of Anoxic-Oxic (A/O) Process for the Changing of Refractory Organics in Electroplating Wastewater: Performance, Optimization and Microbial Characteristics

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 962
Author(s):  
Jingsi Gao ◽  
Chongsen Duan ◽  
Xiao Huang ◽  
Jianghua Yu ◽  
Zhen Cao ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
Performance Optimization ◽  
High Throughput Sequencing ◽  
Microbial Community Composition ◽  
Sodium Dodecyl ◽  
Stable State ◽  
Electroplating Wastewater ◽  
Microbial Characteristics ◽  
Refractory Organics

In order to investigate the tolerance of an anoxic-oxic (A/O) process for the changing of refractory organics in electroplating wastewater, optimize the technological parameters, and reveal the microbial characteristics, a pilot-scale A/O process was carried out and the microbial community composition was analyzed by high-throughput sequencing. The results indicated that a better tolerance was achieved for sodium dodecyl benzene sulfonate, and the removal efficiencies of organic matter, ammonia nitrogen (NH4+-N), and total nitrogen (TN) were 82.87%, 66.47%, and 53.28% with the optimum hydraulic retention time (HRT), internal circulation and dissolved oxygen (DO) was 12 h, 200% and 2–3 mg/L, respectively. Additionally, high-throughput sequencing results demonstrated that Proteobacteria and Bacteroidetes were the dominant bacteria phylum, and the diversity of the microbial community in the stable-state period was richer than that in the start-up period.

scholarly journals Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (5) ◽  
pp. e0197456 ◽  
Author(s):  
Stine H. Kresse ◽  
Heidi M. Namløs ◽  
Susanne Lorenz ◽  
Jeanne-Marie Berner ◽  
Ola Myklebost ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Dna And Rna ◽  
Ffpe Samples ◽  
Rna Extraction Methods

scholarly journals Effects of Andrographolide on Mouse Intestinal Microflora Based on High-Throughput Sequence Analysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Haigang Wu ◽  
Xian Wu ◽  
Li Huang ◽  
Chongmei Ruan ◽  
Jinni Liu ◽  
...  
Keyword(s):  
Microbial Community ◽  
Gut Microbiota ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Bacterial Resistance ◽  
Microbial Community Composition ◽  
Intestinal Flora ◽  
Sequencing Analysis ◽  
Beneficial Bacteria ◽  
Intestinal Microbes

The intestinal flora is a micro-ecosystem that is closely linked to the overall health of the host. We examined the diversity and abundance of intestinal microorganisms in mice following the administration of andrographolide, a component of the Chinese medical herb Andrographis paniculata. Administration of andrographolide produces multiple beneficial effects including anti-inflammatory, antiviral and antibacterial effects but whether it directly influences the gut microbiota is not known. This study investigated whether the oral administration of andrographolide influences the intestinal microbiota and was compared with amoxicillin treatment as a positive control and water only as a negative control. We examined 21 cecal samples and conducted a high-throughput sequencing analysis based on V3-V4 variable region of the 16S rDNA genes. We found that the diversity and abundance of mouse gut microbiota decreased in direct proportion with the amoxicillin dose whereas andrographolide administration did not affect intestinal microbial community structure. The composition of intestinal microbes following andrographolide treatment was dominated by the Firmicutes while Bacteroidetes dominated the amoxicillin treatment group compared with the negative controls. Specifically, the f__Lachnospiraceae_ Unclassified, Lachnospiraceae_ NK4A136_group and Ruminococcaceae_ UCG-014 were enriched with andrographolide administration while Bacteroides, Klebsiella and Escherichia-Shigella significantly increased in the amoxicillin test groups. Amoxicillin administration altered the microbial community composition and structure by increasing the proportion of pathogenic to beneficial bacteria whereas andrographolide administration led to increases in the proportions and abundance of beneficial bacteria. This study provides a theoretical basis for finding alternatives to antibiotics to decrease bacterial resistance and restore intestinal floral imbalances.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

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